Apr 21, 1999 - tion of the bacteria, the histological features of gastritis, and any other pathology such as intestinal metaplasia. The Genta stain has potential ...
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J Clin Pathol 1999;52:693-694
Technical report Modified Genta triple stain for identifying Helicobacter pylori Hala M T El-Zimaity, Jian Wu, David Y Graham Abstract Aim-To evaluate whether lead nitrate could replace uranyl nitrate in the Genta stain for H pylori without sacrificing the advantages of the triple stain (Steiner silver impregnation combined with Alcian blue and haematoxylin/eosin (H&E)). Methods-A comparison was made in 16 specimens between the original triple stain and the revised version. One pathologist evaluated all sections. Results-Direct substitution of lead nitrate for uranium nitrate produced well stained organisms without interfering with H&E or Alcian blue staining. No difference was found in the ability to identify bacteria in 11 cases with H pylori density of 1 or 2 (on a scale of 0 to 5). Conclusions-The potential chemical and radiological hazards associated with uranium nitrate can be eliminated by using lead nitrate without sacrificing the advantages obtained by using the triple stain. (7 Clin Pathol 1999;52:693-694)
Procedure Before staining, place a plastic Coplin jar in a 45-60°C water bath to heat. Prepare the reducing solution and place in the preheated Coplin jar. Steps 1 and 8-18 can be easily perThe Genta stain' is used routinely in our labo- formed on an autostainer. ratory because it allows simultaneous visualisaTo check the accuracy of the modified Genta tion of the bacteria, the histological features of stain for identifying Hpylori, a comparison was gastritis, and any other pathology such as in 16 specimens between the original and intestinal metaplasia. The Genta stain has maderevised One pathologist (HE-Z) the potential disadvantages which include the evaluated allversion. sections. requirement for uranyl nitrate which, in some water. countries, may be difficult or impossible to 1. Deparaffinise and hydrate to distilled in room 2. Sensitise sections placing by obtain. Because of the complexity, time inmastic solulead nitrate-gum temperature volved, and the potential radiological hazard2 of for 30 s. at tion and microwave high power shied have laboratories many nitrate, uranyl in solution for to remain 3. Allow slides away from using the Genta stain. In this comwith a 30 s stir rod). another glass (gently munication, we evaluated whether the widely available lead nitrate could replace uranyl 4. Rinse in distilled water. nitrate in the Genta stain without sacrificing 5. Place sections in 1 % silver nitrate, microwave but do not boil (2 min 15 s). the advantages of the triple stain. 6. Allow slides to remain in the warm solution for 90 s. Methods 7. Wash in distilled water (x4). MODIFIED TRIPLE STAIN 8. Place in a reducing solution and microwave Reagents at high power for 30 s. 1. Gum mastic (Polyscientific), 2.5%; refriger9. Allow slides to remain in the warm solution ate at 4°C. for 3 min or until properly developed. 2. Lead nitrate-gum mastic solution: mix 0.5 g lead nitrate, 40 ml 70% alcohol, and 10 ml 10. Rinse in distilled water (x4). of 2.5% gum mastic. May be reused, but 11. Hydrate back to distilled water. discard after 2 months. Refrigerate at 4°C. 12. Place slides in Alcian blue (10 min).
Keywords: Helicobacter pylori; Genta stain; uranyl nitrate; lead nitrate
Gastrointestinal Mucosa Pathology Laboratory, Veterans Affairs Medical Center and Baylor College of Medicine, 2002 Holcombe Blvd, Houston, Texas 77030, USA H M T El-Zimaity
Department of Medicine, Veterans Affairs Medical Center and Baylor College of Medicine J Wu D Y Graham Correspondence to: Dr El-Zimaity.
Accepted for publication 21 April 1999
3. Silver nitrate, 1%: 0.5 g silver nitrate in 50 ml distilled water. Make fresh each time and filter before use. 4. Silver nitrate, 0.04%: 0.04 g silver nitrate in 100 ml of distilled water. Make fresh each time. 5. Hydroquinone, 2%: 5 g hydroquinone in 250 ml distilled water. Make fresh each time. 6. Reducing solution: mix 100 ml of 2.5% gum mastic, 250 ml of 2% hydroquinone, and 50 ml absolute alcohol. Make just before use, filter through Whatman No 4 filter paper, and add 25 ml of 0.04% silver nitrate. Do not filter after adding the silver nitrate. This solution will have a milky appearance when the gum mastic is added. 7. Alcian blue solution, 1% in acetic acid, pH 2.5 (Polyscientific). 8. Haematoxylin (Gill, triple strength) (StatLab). 9. Treosin (StatLab).
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for sensitisation and reduction are adequate. A comparison between the original and the revised version is summarised in table 1.
enAK Figure I Gastric mucosa showing H pylori using the modified Genta stain.
13. 14. 15. 16. 17. 18. 19. 20.
Rinse in distilled water. Stain in Gill's haematoxylin (5-8 min). Rinse in tap water. Quickly dip in ammonia water or lithium carbonate (0.5%). Wash in tap water (two changes). Stain in eosin (five min). Wash in tap water. Dehydrate through graded alcohols and clear in xylene.
Results Direct substitution of lead nitrate for uranium nitrate produced well stained organisms without interfering with haematoxylin and eosin or Alcian blue staining (fig 1). Eleven cases with H pylori density of 1 or 2 (on a scale of 0 to 5)3 were examined. No difference was found in the ability to identify the bacteria. The staining time was also reduced by 46 minutes by the eliminating the gum mastic step between silver nitrate impregnation and reduction and by using the microwave oven at sensitisation, silver impregnation, and in the reduction step. Because of the variation in microwave wattage, timing in the microwave will vary. Generally, 2 minutes 15 seconds for silver impregnation and 30 seconds Table 1 Comparison between the original and the revised version of the Genta stain Procedure
Original Genta
Modified Genta
Deparaffinise and hydrate Sensitise sections Slides remain in solution Rinse in distilled water 1% silver nitrate
20 s 3 min
20 st 30 s 30 s 5s 2 min, 15 s 1 min, 30 s 5s
Slides remain in solution Wash in distilled water Rinse in 95% and 100% alcohol (x2 each) Gum mastic Reducing solution Slides remain in solution Rinse in distilled water Hydrate back to distilled water Place slides in Alcian blue Rinse in distilled water Gill's haematoxylin Rinse in tap water Ammonia water Tap water Eosin Wash in tap water Dehydrate and clear in xylene
Totaltime
tSteps that can be performed on an autostainer.
5s 2 min, 15 s 10 min 5s
10 s 10 min 25 min
Eliminated Eliminated 30 s
5s
3 min 5 st 10 st
10 s 10 min 5s 7 min 5s 2s 2s 5 min 2s 10 s 74 min, 8 s
10 mint 5 st 7 mint 5 st 2 st 2 st 5 mint 2 st
10 st 28 nmin, 4 s
Discussion The Genta stain' combined the modified Steiner method4 with Alcian blue and haematoxylin and eosin to facilitate the examination of Hpylori and its associated pathology such as gastritis and intestinal metaplasia on the same slide. While the stain is used routinely in our laboratory, it did not gain acceptance in many laboratories owing to the lengthy staining time and the use of uranyl nitrate with its potential radioactive hazards. Steiner's method requires the use of heat to impregnate tissue with silver nitrate. Subsequently, slides are placed in a reducing solution that converts silver nitrate to black metallic silver deposits on the bacteria. Garvey and colleagues4 reduced the staining time with a developing solution that was easier to prepare than the original Steiner formula.' Swisher6 used the microwave oven at sensitisation and silver impregnation. Finally, Churukian further reduced the staining time by also performing the reduction step in the microwave oven rather than in a hot water bath (Churukian C, personal communication). In 1979, Elias et al reported that it was possible to use lead nitrate as a substitute for uranyl nitrate in the Steiner stain and thus avoid the potential radiation hazards of uranyl nitrate.7 We incorporated these different suggestions4'6 7in the current modification of the Genta triple stain. The use of lead nitrate-gum mastic solution and the microwave oven at the sensitisation, silver impregnation, and reduction steps reduced the staining time by 264%. In addition, deparaffinisation (step 1) and all steps following reduction (step 9) can be done with the help of an autostainer. Lead nitrate provides a substitute that is widely available, free of any potential radiation hazard, and without loss of staining specificity. The technique is thus quicker than before, simple to perform, and possible to undertake in any laboratory. Using an autostainer, the total technical time is only 9-10 minutes. 1 Genta RM, Robason GO, Graham DY. Simultaneous visualization of Helicobacter pylori and gastric morphology: a new stain. Hum Pathol 1994;25:221-6. 2 Darley JJ, Ezoe H. Potential hazards of uranium and its compounds in electron microscopy: a brief review. J Microsc 1976;106:85-6. 3 El-Zimaity HM, Graham DY, Al-Assi MT, et al. Interobserver variation in the histopathological assessment of Helicobacter pylori gastritis [see comments]. Hum Pathol
1996;27:35-41.
4 Garvey W, Fathi A, Bigelow F. Modified Steiner for the demonstration of spirochetes. J Histotechnol- 1985;8:15-17. 5 Steiner G, Steiner G. New simple silver stain for demonstration of bacteria, spirochetes, and fungi in sections of paraffin embedded tissue blocks. Jf Lab Clin Med 1944;29:86871. 6 Swisher BL. Modified Steiner procedure for microwave staining of spirochetes and nonfilamentous bacteria. Jf Histotechnol 1987;10:241-3. 7 Elias JM, Greene C. Modified Steiner method for the demonstration of spirochetes in tissue. Am Jf Clin Pathol 1979;71:109-1 1.
